Abducted as an unnamed infant by Orochimaru, the future Yamato became one of sixty children subjected to DNA experiments with Hashirama Senju's cells to replicate Wood Release and tailed beast control. He alone survived, gaining Wood Release abilities. Orochimaru abandoned the project, believing all had died, but the child escaped and was found by Danzō Shimura. Recruited into Root, Konohagakure's covert Anbu, as "Kinoe," he received training from Danzō, who provided scrolls to refine his Wood Release.

Danzō ordered Kinoe to assassinate Third Hokage Hiruzen Sarutobi. Kakashi Hatake, then in Root, intervened, defeated Kinoe, but spared him, prompting Kinoe to question Root's methods. Later, Danzō sent Kinoe to aid Orochimaru's escape via the smoke-wielding Iburi clan. There, he met Yukimi, who mistook him for her deceased brother "Tenzō." After Orochimaru killed the clan and captured Yukimi, Kinoe and Kakashi rescued her. Yukimi survived, and Kinoe adopted the name Tenzō in her brother's memory, solidifying his rejection of Root.

Danzō next ordered Tenzō to steal Kakashi's Sharingan. During the mission, Tenzō defied Danzō after Kakashi reaffirmed comradeship's value. Kakashi saved Tenzō from Danzō's retaliation, leading Third Hokage Hiruzen to transfer him to regular Anbu under Kakashi in Team Ro. He served with distinction, achieving the highest mission success rate under Hiruzen and witnessing the Uchiha massacre aftermath alongside Itachi Uchiha.

Years later, Fifth Hokage Tsunade assigned him to temporarily lead Team Kakashi during Kakashi's absence, giving him the codename "Yamato." His Wood Release uniquely suppressed the Nine-Tails within Naruto Uzumaki. He managed team dynamics, using an intimidating "ghoul-eyes" expression to quell Naruto's disobedience. Key missions included infiltrating Orochimaru's hideout to retrieve Sasuke Uchiha and gathering intelligence on Sasori via Kabuto Yakushi.

After Pain destroyed Konoha, Yamato used Wood Release to expedite reconstruction. During the Fourth Great Ninja War, Kabuto Yakushi captured him to harvest Hashirama's cells for White Zetsu clones. Freed post-war, he was reassigned to monitor Orochimaru, a role continuing into the Boruto era.

Yamato exhibits a calm, stoic demeanor on missions but dry humor off-duty. He integrates his architecture hobby into Wood Release, often creating structures like houses. He insists on his current "Yamato" codename over "Tenzō," though Kakashi occasionally uses the latter, and emphasizes mission preparedness. His techniques include Wood Release: Four Pillar House Technique for shelter, Wood Release: Serial Pillar Houses for combat, and Hokage-Style Sixty-Year-Old Technique to suppress tailed beasts.